PROJECT SUMMARY ! UHRF1 (ubiquitin-like, PHD and RING finger containing 1) is a multi-domain E3 ubiquitin ligase overexpressed in many human cancers. Deregulation of UHRF1 has been shown to correlate with cell proliferation and metastasis, and loss of UHRF1 renders cancer cells hypersensitive to DNA damaging agents. Genetic studies determined that UHRF1 is required for the epigenetic inheritance of DNA methylation, and that recognition of methylated histone H3 lysine 9 by the UHRF1 tandem Tudor domain (TTD) and adjacent plant homeodomain (PHD) is necessary to maintain DNA methylation patterns in human cancer cells. UHRF1 also contains a SET and Ring-associated domain (SRA) that binds hemi-methylated DNA, suggesting UHRF1 may recruit DNMT1 to replicating chromatin. Collectively, these data demonstrate that UHRF1 plays a fundamental role in the DNA methylation program through multivalent engagement of epigenetic modifications on the chromatin template. Because DNA methylation and histone PTMs are not coded for in the DNA sequence, vary among cell types, and are mediated in a large part by ?druggable? effector proteins, targeted intervention of proteins that recognize and interpret these epigenetic modifications represents a novel and promising cancer therapeutic strategy. We hypothesize that chemical disruption of the binding interaction between UHRF1 and chromatin is a rationale therapeutic approach to modulate cellular DNA methylation in human cancers. The goal of this proposal is therefore is to discover potent, selective, cell-active antagonists of the histone and DNA binding domains of UHRF1. These studies will set a strong foundation for subsequent drug discovery platforms targeting complex epigenetic readers and will facilitate the future study of UHRF1 function in normal biology, as well as developmental and disease states.